Expert Review Panel for Kombucha Tea

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Expert Review Panel

for Kombucha Tea

ATLANTA MARRIOTT MARQUIS S E P T EMB E R 24 , 2 0 1 6 1 : 0 0 PM – 4 : 0 0 PM R O OM : M101

contact: spsfam@aoac.org

AOAC Stakeholder Panel on Strategic Food Analytical Methods Ethanol in Kombucha Expert Review Panel

Sunday, September 24, 2017, 1 :00 p.m. – 4:00 p.m. Atlanta Marriott Marquis, Room M101

A G E N D A

1. Welcome and Introductions Sneh Bhandari, Mérieux NutriSciences (ERP Chair)

2. Review of AOAC Volunteer Policies & ERP Proccess Overview and Guidelines Deborah McKenzie, AOAC INTERNATIONAL

3. Review of Methods For each method, the assigned ERP members will present a review of the revised method manuscripts, after which the ERP will discuss the method and render a decision on the status for each method.

A. KOM-04

a. Mirzoian Review b. Bhandari Review c. Discussion and Vote

B. KOM-05

a. Ebersole Review b. Jayabalan Review c. Discussion and Vote

C. KOM-06

a. Bhandari Review b. Ebersole Review c. Discussion and Vote

D. KOM-07

a. Joseph Review b. Mirzoian Review c. Discussion and Vote

E. KOM-08

a. Stenerson Review b. Jayabalan Review c. Discussion and Vote

4. Final Action Requirements for Approved Method(s) 5. Adjourn

SPSFAM Kombucha ERP 09/15/2017 – v1.0

AOAC SPSFAM KOMBUCHA EXPERT REVIEW PANEL

METHODS AND SMPR ACCESS

• AOAC SMPR 2016.001 • METHODACCESS [ERP ONLY - PASSWORD REQUIRED]

AOAC Stakeholder Panel on Strategic Food Analytical Methods: Expert Review Panel AOAC Candidate Method #KOM-04 (Resubmission) Single-Laboratory Validation of the Determination of Ethanol in Kombucha by Gas Chromatography-Flame Ionization Detector: Intra-Laboratory Validation

• Author(s): Xin Du and Yonglin Ren • Submited by: Xin Du, Murdoch University • Attachments: 0

Submitter notes: None.

Primary Reviewer: Armen Mirzoian, US Alcohol & Tobacco Tax & Trade Bureau Secondary Reviewer: Sneh Bhandari, Mérieux NutriSciences

• Author(s): Xin Du and Yonglin Ren • Submited by: Xin Du, Murdoch University • Attachments: 0

Submitter notes: None.

Primary Reviewer: Armen Mirzoian, US Alcohol & Tobacco Tax & Trade Bureau Secondary Reviewer: Sneh Bhandari, Mérieux NutriSciences

AOAC SPDS ERP REVIEW: MAIN FORM

Submission Date

2017-09-18 09:24:21

Name

Armen Mirzoian

E-mail

armen.mirzoian@ttb.gov

Organization

TTB

Title of Method

Determination of Ethanol in Kombucha by GC-FID

AOAC Candidate Method Number (e.g. ALN-01)

KOM-4

Applicable SMPR

2016.001

Summary:

The method measures concentration of ethanol in Kombucha by liquid injection GC- FID using 2-propanol as internal standard

1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used. 4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference.

Generally it does, however it's not clear how are the LOQ and LOD evaluated.

No 1. Since determination of LOQ and LOD is not described, it is not clear whether they meet SMPR. 2. Recoveries for ethanol concentrations below 0.5% abv were not determined 3. Repeatability of the method (presented as Precision) exceeds 4% 4. Reproducibility was not determined

1. LOQ and LOD are neither defined nor evaluated. 2. Spike recovery determination is incomplete 3. Precision is only expressed as repeatability 4. Reproducibility was not determined

yes

Same as II.3. 1. LOQ and LOD are neither defined nor evaluated. 2. Spike recovery determination is incomplete 3. Precision is only expressed as repeatability 4. Reproducibility was not determined

2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. 3. Is there information demonstrating that the

No. Only VWR ethanol (99.96% abv putiry) was used.

No. Same as I.3 1. Since determination of LOQ and LOD is not described, it is not clear whether they meet SMPR. 2. Recoveries for ethanol concentrations below 0.5% abv were not determined 3. Repeatability of the method (presented as Precision) exceeds 4% 4. Reproducibility was not determined

Only partial information on system suitability tests can be found in Tables 1 and 2. However, they were not intended or described as such.

Only partial information on the method's suitability tests can be found in Tables 1 and 2. However, they were not intended or described as such.

Yes, it's clear and concise however fair amount of required information is missing

5. Based on the supporting information, what are the pros/strengths of the method? 6. Based on the supporting information, what are the cons/weaknesses of the method?

GC-FID is widely available in many analytical laboratories; although the sample preparation step is not described, most likely it's dilute and shoot.

Potentially long analyses time (GC program is not described); small kombucha producers may not have access to GC-FID

7. Any general comments about the method?

In addition to the comments above:

1. The text states only two calibration standards at 0.1% and 0.5% abv. 2. Simple calculations of the calibration standard preparation procedure gives 5% and 25% abv solutions, however 0.1% and 0.5% abv is stated 3. It is not clear how calibration standards in the measured 0.1%-2% abv range were prepared. 4. Specificity and robust of the method was not addressed?

Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

AOAC SPDS ERP REVIEW: MAIN FORM

Submission Date

2017-09-12 16:28:27

Name

Sneh BHANDARI

E-mail

SNEH.BHANDARI@MXNS.COM

Organization

Merieux NutriSciences

Title of Method

Determination of ethanol in Kombucha by Gas Chromatography-Flame Ionization Detector: Intra-Laboratory Validation

AOAC Candidate Method Number (e.g. ALN-01)

KOM-04

Applicable SMPR

AOAC SMPR 2016.001

Summary:

Low level of ethanol is detected in Kombucha by gas chromatography with flame ionization detection employing 2-propanol as an internal std. Applicable for determination of 0.1-2% ABV ethanol in Kombucha tea beverage. LOD and LOQ = 0.003 and 0.011 %ABV respectively. The method precision as RSDr was found to be 0.55-2.5% and % spike recovery in the range of 99.-99.5% at different spike levels.

Yes

No. The precautions in use of flammable solvents in the method may be stated. The precautions and warning in use of gases under pressure may also be mentioned.

1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method.

Yes

The SLV data don’t provide any accuracy evaluation of the method using any reference materials. Spike recovery evaluation to some extent may support the accuracy of the method. The precision data are provided for 10 injections of low-level in house sample. This is precision of injection not the analysis. The authors did apply the method to the commercial samples. Information is not available to support the precision of the method in analysis of ethanol in Kombucha tea samples. No information available about the evaluation of the method accuracy employing the Certified reference materials, The analytical range of the method not specified.

No information available about the evaluation of the method accuracy employing the Certified reference materials,

The analytical range of the method not specified.

3. Is there information demonstrating that the

The SLV data support the accuracy of the method by spike recovery evaluation. But it doesn’t provide whether spike recovery was performed in Kombucha product or in ethanol in water. It doesn’t specify how many replicates were analyzed at every spike level. It seems only single replicate was analyzed at three spike level. Provided % Recovery data meets SMPR requirements. The precision data are provided for 10 injections of low-level in house sample. This is precision of injection not the analysis. The authors did apply the method to the commercial samples. Information is not available to support the precision of the method in analysis of ethanol in Kombucha tea samples

method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified.

Data about the analytical range, LOD and LOQ are not available.

The SLV data don’t provide any accuracy evaluation of the method using any reference materials. Spike recovery evaluation to some extent may support the accuracy of the method

Yes. Precaution in use of flammable solvents and gases under pressure may be mentioned.

The method does not indicate any system suitability criteria. The method may indicate resolution requirement of ethanol and internal std peak as they elute very close to each other. The method may also indicate requirement of repeat-ability of the lowest calibrant and the ruling out any interference from the method blank.

3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. 5. Based on the supporting information, what are the pros/strengths of the method? 6. Based on the supporting information, what are the cons/weaknesses of the method?

The method does not indicate any system suitability criteria.

The method as written seems not be complete. It does not provide details about how the samples are prepared for analysis. The details of the amount addition of internal std. in the sample and calibrartion std. missing.

The supporting data don't provide complete details about the method, i.e., analytical range of the method, what samples were used for the spike recovery evaluation etc.

Simple and easy method to operate. Spike recovery satisfactory (but sample details missing).

1. No data available to know the method precision in analysis of ethanol in Kombucha Tea. 2. The method provide no information to rule out interference from the components of Kombucha tea in accurate and precise quantitation of ethanol in samples. 3. The method accuracy not established using Certified ref materials. 4.Information not available to rule out the possibility of carry over from injection to injection. 5.Retention time of internal std and ethanol are close. The available data on the method performance don't rule out that this does not impact the accuracy of the method particularly in ethanol estimation at the low levels. 6. Information about the Analytical Range of the method missing.

7. Any general comments about the method?

The method performance evaluation is not yet complete. The method as written is not complete and missing many of the critical

Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

Not without getting additional details about the missing information in the method and its performance evaluation.

AOAC Stakeholder Panel on Strategic Food Analytical Methods: Expert Review Panel AOAC Candidate Method #KOM-05 (Resubmission) Determination of Alcohol Content in Kombucha Tea by Headspace Solid Phase Microextraction and Gas Chromatography-Mass Spectrometry • Author(s): Katherine Stenerson • Submitted by: Katherine Stenerson, MilliporeSigma • Attachments: 0 Submitter notes: The following have changed to include the validation data required: Table 2: Validation across analytical range in Kombucha tea matrix Table 3: Method LOD and LOQ, calculated in Kombucha tea matrix Table 4.: CRMs, 2 analysts, 2 instruments Table 5: added 1 additional Kombucha sample (#13) Figure 3: Comparison of method linearity from water vs. Kombuchalacke E Reviewers: Primary Reviewer: Blake Ebersole, NaturPro Scientific, LLC Secondary Reviewer: Rasu Jayabalan, National Institute of Technology, Rourkela, India

AOAC Kombucha ERP Reviewer: Blake Ebersole KOM-05 Stenerson method review – HP-SPME-GC-MS

Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. * The method is a headspace GC-MS method with a solid phase microextraction step. The method is expected to be applicable for the SMPR. Headspace sampling for volatile substances like ethanol is a proven way to reduce matrix interference versus direct injection methods. Solid Phase Microextraction is a newer way to reduce the likelihood of matrix interferences. GC-MS is highly sensitive in its separation and detection of pure compounds from complex mixtures. Potential limitations of the method include a potential for incomplete extraction of analyte through the microextraction step, and the saturation and non-linearity of the MS detector at higher concentrations. 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. * The instrumentation used would be expected to meet these SMPR for ethanol in kombucha. The method includes a number of separation and purification steps that are advanced compared to what has been traditionally used for alcohol in beverages in terms of sensitivity and eliminating potential matrix interferences. The authors also used deuterated ethanol as an internal reference standard to more accurately determine recovery of spiked ethanol in fermented samples. A dilution factor of 10:1 was applied across all standards and samples to correct for potential linearity issues due to detector saturation at higher concentrations. The study materials included certified reference materials for beer and ethanol/water. The study materials used to calculate method repeatability (%RSD) did not include kombucha that contains naturally high amounts of alcohol, but did use a low-alcohol kombucha spiked with known amounts of pure ethanol. It is possible that the matrices represented in the repeatability analysis do not reflect the range of possible types of kombucha as defined in the SMPR. For example, a higher alcohol, unfiltered kombucha may have different amounts of solids like yeast and bacteria which can be matrix interferences. The authors did perform duplicate analysis on 13 commercial kombucha samples, of which most were high alcohol. Percent differences on duplicate measures of these samples were calculated, that can be used to support the repeatability data. There were also several ruggedness tests performed that are not required by many validation guidelines. The use of different microextraction columns, GC column temperatures, and diluents were studied to determine their impact on linearity and precision. The authors also used a deuterated ethanol internal standard which can help to distinguish between naturally occurring alcohol and a reference spike. 3. Terminology –

In most cases, terminology is used appropriately. In some cases, the term reproducibility is used interchangeably with repeatability. In this study, reproducibility is defined as two instruments and analysts, and it was not clear whether these were in different labs with different sample prep/procedures areas, or within the same lab.

4. Safety precautions - No information was given on safety precautions.

Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. * Definitions are used appropriately 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. * The information demonstrates that the method is appropriate for the reference materials ethanol. The percent recovery was between 98 and 100%, within the SMPR of 97% to 102%. The analytical range for the method was the same as in the SMPR, 0.1% to 2.0%. The LOQ for buffer spiked with ethanol was 0.03% ABV, lower than the SMPR LOQ of 0.05%. %. The RSD of 4 spiked samples were between 1-3%. The mean % difference of 13 duplicates of high alcohol kombucha was 5.1%, similar to the RSD of 4%. Reproducibility was also presented as the same method using certified reference materials (beer and ethanol-water) on different instruments and different analysts, and the RSD was 3-5%. 3. Is there information demonstrating that the method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. * I am not sure whether the repeatability calculation given for the higher alcohol kombuchas is sufficient, since it is not the result of a standard deviation calculation. I would be open to discussion on this part. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? * Based on the ruggedness data, I would recommend to include ruggedness data within the method, along with a reminder that changes to the method should be re-validated as part of internal method acceptance procedures.

2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. * A number of controls were provided. Method performance results for linearity, LOQ, accuracy and precision were provided. Reference chromatograms and spectral data was not included.

3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. * Yes

4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. * The method was written clearly and concisely.

5. Based on the supporting information, what are the pros/strengths of the method? * The method is highly sensitive and selective for ethanol. It also uses modern technology to reduce the chances for matrix interference. It also uses a constant dilution factor within the analytical range, and deuterated ethanol internal standards, to be able to separately distinguish the internal standard from naturally occurring ethanol.

6. Based on the supporting information, what are the cons/weaknesses of the method? * As mentioned, the repeatability study was done in a low-alcohol kombucha matrix spiked with a known amount of ethanol. The precision results from this study were excellent. No formal repeatability was done on higher kombucha samples. Regardless of alcohol content, a case can be made that one sample from one manufacturer does not reflect the diversity of manufacturing processes used for kombucha. Consequently, higher imprecision was observed in the naturally high alcohol commercial samples, with the percent difference of duplicates being greater than 4% for 6 of 13 samples, and greater than 10% for 2 of these. Repeatability RSD was not performed on other kombucha samples that contained higher amounts of alcohol, without spiking. If a high alcohol kombucha was included in the repeatability analysis, it would be easier to rule out the potential interferences that may not be accounted for by spiking a low alcohol kombucha. The method could also have provided additional system suitability parameters such as reference chromatograms or spectral data for quality control purposes. One complaint often made by traditionalists of alcohol analysis is the cost and expertise required for newer and more advanced methods, of which is this one example. However, the robustness of the method, expertise require and the analysis time should be balanced against cost.

7. Any general comments about the method? * This is an accurate and precise method and I praise the authors for the work and use of novel instrumentation to address some issues for this challenging matrix. The data presented are intriguing because it also shows how slight changes in the method cause results to fall outside SMPR. For example, the internal standard (deuterated ethanol) diluted in water versus a salt-buffer solution had a repeatability of 9%, higher than the SMPR of 4%. Decreasing column temperature by 10 degrees led to a repeatability of 9%, versus 0.9% reported with the optimized method. The data suggests that there are good reasons to re-validate a method whenever slight modifications are made.

Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale. * I recommend the method be adopted as First Action due to the robustness of the method. I would also recommend additional precision data on higher alcohol kombucha samples to be generated.

AOAC SPSFAM ERP REVIEW: MAIN FORM

Submission Date

2017-09-16 22:05:30

Name

Rasu Jayabalan

E-mail

jayabalanr@nitrkl.ac.in

Organization

National Institute of Technology, Rourkela, Odisha, India

Title of Method

Determination of alcohol content in kombucha tea by head space solid phase microextraction and gas chromatography-mass spectrometry

AOAC Candidate Method Number (e.g. ALN-01)

KOM-05

Applicable SMPR

Ethanol in Kombucha Tea (2016.001)

I. Summary of the Method

This is a head space - solid phase microextraction (HS-SPME) GC-MS developed for determination of alcohol in Kombucha tea. The method describes the reagent preparation, instrument details and validation of the method using GC-MS. Suitable standards and samples were used. Dilutions need to be followed are explained. The method detects and quantitate the ethanol within the prescribed limits of LOD and LOQ. However the role of Mass spectrometry is not clearly mentioned.

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used. 4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s).

Yes. The method has quoted the SMPR parameters whereever applicable.

Yes. The method meet the SMPR parameters like LOD, LOQ, and Reproducibility. All the parameters checked are within the prescribed limits given in SMPR.

Yes. The method has quoted the SMPR parameters and applied them appropriately to validate the method.

Ethanol, sodium chloride, sodium potassium phosphate (monobasic and dibasic) are used in the method. Hazardous reagents were not used in this method.

III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. 3. Is there information demonstrating that the

Yes. The definitions specified in the SMPR were not defined in the document, but they have been appropriately applied.

Yes. The method meets the SMPR method performance requirements like LOD, LOQ, reproducibility.

Yes. The method performs withing the SMPR method performance requirements table specifications for the ethanol.

Supporting information: There are no supporting documents provided with the main document. All the steps in method evaluations have been explained in the main document.

Yes. The method has utilized SMPR recomended 200 proof ethanol (99.5%) and Low alcohol beer (nominal 0.50% ABV) from Millipore Sigma as standard.

Yes. the method is written clearly and concisely.

Strengths: 1. The method is able to accurately detect the ethanol concentration as low as 0.03%.

Weakness: 1. Since the method needs GC-MS, small analytical laboratories will not be able to use the method. 2. Method needs highly skilled technicians. 3. Method is considered to be expensive due to the use of GC-MS. The method and results given in the manuscript is able to quantitate the ethanol as per SMPR specifications. The method used recommended standards and samples. The method explains the dilution protocol need to be followed. Skilled technicians would be able to follow the method clearly. Yes. I would recommend this method be adopted as a First Action and publish in the Official Methods of Analysis of AOAC. Rationale: The methodology was developed using standards and samples recommended by SMPR and is able to detect and quantitate ethanol level prescribed in the SMPR. The method performance requirements given in SMPR is applicable.

7. Any general comments about the method?

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

AOAC Stakeholder Panel on Strategic Food Analytical Methods: Expert Review Panel AOAC Candidate Method #KOM-06 Enzymic method for measurement of Ethanol in Kombucha • Author(s): R. Ivory, E. Delaney, J. Larkin, V. McKie and B. V. McCleary

• Submitted by: Ruth Ivory, Megazyme

• Method applicability: The method developed meets the standard method performance requirements that can be tested in a single lab. It is currently the best method available for enzymatic testing of ethanol in Kombucha products.

• Attachments: 0

• Submitter notes: None

Primary reviewer: Sneh Bhandari, Mérieux NutriSciences Secondary Reviewer: Blake Ebersole, NaturPro Scientific LLC

AOAC SPDS ERP REVIEW: MAIN FORM

Submission Date

2017-09-12 15:56:34

Name

SNEH BHANDARI

E-mail

SNEH.BHANDARI@MXNS.COM

Organization

Merieux NutriSciences

Title of Method

Enzymatic method for measurement of Ethanol in Kombucha

AOAC Candidate Method Number (e.g. ALN-01)

KOM-06

Applicable SMPR

2016.001

Summary:

Enzymatic method for measurement of ethanol in Kombucha. The method is based on Megazyme’s Ethanol-assay kit (K-ETOH).

The quantification of ethanol requires two enzyme reactions; in the first reaction catalysed by alcohol dehydrogenase (ADH), ethanol is oxidised to acetaldehyde by nicotinamide-adenine dinucleotide (NAD+) (1) Ethanol + NAD+ ADH ------------  acetaldehyde + NADH + H+ However, since the equilibrium of reaction (1) lies in favour of ethanol and NAD+, a further reaction is required to “trap” the products. This is achieved by the quantitative oxidation of acetaldehyde to acetic acid in the presence of aldehyde dehydrogenase (Al-DH) and NAD+. (2) Acetaldehyde + NAD+ + H2O Al-DH-----------------  acetic acid + NADH + H+ The amount of NADH formed in this reaction pathway is stoichiometric with twice the amount of ethanol. It is the NADH which is measured by the increase in absorbance at 340 nm.

Yes

4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method.

Yes

There are no data available to establish accuracy/trueness of the submitted method using some certified or standard reference materials (SRM) with known ethanol concentrations. It is possible because currently there are commercially available SRM for Kombucha Tea matrix. It will help if the method accuracy could be demonstrated using other relevant reference materials like BCR 651 (Beer, ethanol = 0.505%), BCR 652 (Beer, ethanol = 0.051%).

3. Is there information demonstrating that the

2.2 Analytical Range and limit of quantification (LOQ) The authors have nicely demonstrated linearity of the assay. LOQ Calculations by the authors: 1. The authors may clarify their calculations of LOQ systematically and clearly. Also back calculate the expected mcg of analyte in assay with the calculated LOQ . 2. Table 6 provides data for calibration for ethanol stock solution of 0.065% but not for 0.0395% claimed LOQ of the method. This probably requires further clarification. Recovery Data: 1. The authors have presented their spike recovery data in Tables 1-4, for 4 different Kombucha sample studied. Can results in Tables 1-4 presented as % Ethanol (v/v) so that it’s easy to compare the data across the study. 2. The assay is designed to run a standard (0.05 mg/mL) in batch of the analysis. The authors may clarify the purpose of running standard in the assay. It’s not used in calculation. The calculation employs NADH extinction coefficient in calculation of ethanol depending on number of molecules of NADH generated in the assay. Is it possible demonstrate accuracy of the assay by calculation of ethanol using ΔA values obtained for the standard. 3. The assay analyzed standard in every batch. Is its purpose to monitor the assay performance for the batch? The range of ΔA values (0.5 + ??) may be helpful if the purpose of running standard mentioned in the earlier sentence is correct. 4. It may be pointed out the that ΔA values observed for two of the samples in the study is lower than that obtained for the corresponding standard in the batch. 5. The authors have demonstrated a satisfactory recovery of ethanol in four of the studied Kombucha samples based on a single spike level (Kombucha + Ethanol standard 0.05 mg/mL). Listed % spike Recovery is satisfactory 97.7-101.6%. It will be nice to know that the assay provide satisfactory spike recovery at multiple spike concentrations also. 3.3 Precision/ Repeatability Evaluation: 1. The precision (RSDr) was determined by analysis of 3 replicates in a single day and was found to be satisfactory % RSDr = 0.14-1.63%. 2. The precision of the assay performed over two days by two analyst (total number of replicates n = 4) provided % RSD in the range of 3.1 - 5.2%. This value is higher than Repeatability (RSDr) requirement of the SPMPR (2016.001) which is < 4% and lower than Reproducibility (RSDR) requirements of the SMPR which is < 6%. Precision of the assay as %RSD by different technicians on two different days increased 2-3 fold in comparison when it’s performed by a single analyst in a single day. It is likely that the reproducibility % RSD of the assay observed by the method developers within lab will increase further when the assay is performed by different labs. Thus based on the current data there is a good possibility that the method reproducibility across different laboratories is likely to be above SMPR requirements of < 6%.

1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify.

Not very clearly

System suitably not clearly defined

4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions.

The Following clarification about method will help

1.8 Sample Preparation: c. Samples diluted 1:200 with distilled water. The authors may state how the dilution was performed (and recommended ), any minimum volume of Kombucha need to be pipetted to get good precision data?. d. Analyze the samples as per the Megazyme Ethanol Assay Kit (K-ETOH) (We were not provided with a copy of the instructions). Is the method in the kit instructions different that listed here. “-------, lengthen the incubation time to 10 minutes to ensure completion of the reaction”. Does the K-ETOH kit instruction mention a shorter incubation time ? Is it expected for the analysts to watch every sample for about 15 minutes to confirm that reaction has stopped in 10 minutes? The authors may provide some guidelines. 1.9 Manual Assay procedure Was the assay performed directly in curettes or certain size of tubes? How were solutions mixed in the container of the reaction before incubations? – whether hand shaking or vortex or some other way? How did you manage the incubation time? – whether fixed 10 minutes or checked it in every case? 2.1 Selectivity, accuracy and interference: “If the conversion of ethanol has been completed within time specified in the assay (approx. 10 minutes), it can be generally concluded that no interference has occurred”. Authors may provide data or justify this statement specific to this method and matrix in scope. “However, this can be further checked by adding ethanol (about 5 µg in 0.1 mL) to the cuvette on completion of reaction”. Please clarify this further and whether authors had an opportunity to perform the mentioned experiment to establish selectivity of the assay. It may be important to establish selectivity of the method. It will help to know that some of the components which can be in Kombucha like, glucose, fructose, sucrose, gluconic acid, acetic acid, lactic acid and citric acid , caffeine etc. don’t interfere in the ethanol assay by this method. Stability of the Kit: The authors claim the shelf-life of the most of the components of the kit is >1 year at 4 °C. The authors may provide data to support this claim. It's an easy, simple and very promising method for the matrix. Establishing Selectivity and accuracy of the method will help further validate the method for the targeted application. The method is quite promising but some additional information as specified earlier about the method selectivity and accuracy is required for further recommendation of the method for First Action. Simple assay

5. Based on the supporting information, what are the pros/strengths of the method? 6. Based on the supporting information, what are the cons/weaknesses of the method?

7. Any general comments about the method?

Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

AOAC Kombucha ERP Reviewer: Blake Ebersole KOM-06 Ivory et al. method review – Megazyme kit Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. * The method is an enzymic method to determine ethanol in kombucha, based on the conversion of ethanol to acetaldehyde, and then to acetic acid. The method is an indirect method, but is widely used for analysis of ethanol content, and is expected to be applicable for the SMPR 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. * The techniques shown do not meet the SMPR due to the highest concentration sample tested being approx 20% lower than 2% ABV, the maximum end of the analytical range. 3. Terminology – In most cases, terminology is used appropriately. In some cases, the term reproducibility is used in place of ‘intermediate reproducibility’ or ‘within-lab reproducibility’, In this study, reproducibility is defined as two instruments and analysts, in the same laboratory. 4. Safety precautions – References to safety information was referred to SDS and operators manual. Storage conditions for kombucha samples were not noted. Suggest to add a precaution regarding keeping samples refrigerated and the handling of carbonated samples in glass bottles, if applicable. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. * Definitions are used appropriately

No standard reference materials or certified reference materials listed in the SMPR were used. 200 proof ethanol from Sigma, listed in SMPR was diluted for the calibration curve and spike. Lack of use of standard or certified reference materials could impact the robustness of the method. Only 4 kombucha samples from 2 manufacturers were included in the study

3. Is there information demonstrating that the method performs within the SMPR Method Performance Requirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. * There is information demonstrating the method performs to SMPR, with some gaps: 1. Range of analyte concentrations from kombucha did not bracket the required analytical range. The highest concentration of kombucha measured at 1.6%, lower than the 2% listed in the SMPR. 2. The recovery of ethanol in commercial kombucha was reported in table 5 as approximately 0.1, 0.5, 0.5 and 0.7. These values differ from those in table 8 on the same brands (0.3-1.6 % ABV). The differences conflict with the recovery data which is very tight. Request some explanation. IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? * Some of the controls on the assay procedure were not entirely clear (see following section) 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. * --It was stated that ‘approximately 2 mL’ is taken and filtered and vortexed before dilution. The method indicated an ‘appropriate’ dilution should be made, in this study it was a 1:200 dilution. There was no indication of whether the 1:200 or other appropriate dilution should be made volumetrically, and the precision or calibration of the volumetric transfer. -- The controls for determining the end of the reaction lacked some clarity: Sec 1.9 of procedure reads: “Mix and read the absorbances of the solutions at the end of the reaction (Approx 10 minutes). If the reaction has not stopped after 10 minutes, continue to read the absorbances at 1 minute intervals until the absorbances increase constantly over 5 minutes**. ** If this creep rate is greater for the sample than for the blank, extrapolate the sample absorbances back to the time of addition of suspension 4.” Could we request some clarification on how the analyst is to know the endpoint of the reaction, and what does a ‘constant increase’ in absorbance look like? -- The method did not determine whether acetic acid or acetaldehyde expected to be found naturally in kombucha samples (and which are reactants in the enxymatic reaction) might interfere with the reaction or the results. --The method states that cross-reactivity with methanol is not an issue, but did not present data demonstrating this.

-- The method states that blank cuvettes can either be filled with air or water, but did not present data supporting the validity of both.

3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. * Yes

4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. * The method was written clearly and concisely.

5. Based on the supporting information, what are the pros/strengths of the method? * The method is highly selective for ethanol, as an enzymatic method . It is an inexpensive and rapid method. The repeatability and recovery data were very strong.

6. Based on the supporting information, what are the cons/weaknesses of the method? * Indirect, enzymatic methods can be prone to interferences, although if there were any in this study, they were not obvious from the data.

The range of analyte concentrations did not bracket the analytical range in the SMPR

The method of preparing the sample and monitoring the reaction may have lacked sufficient clarity.

No SRM or CRM were used in the study

Only two manufacturers, from UK, and 4 products were represented in the set of kombucha matrices

No ruggedness data was presented. Changing reaction times might have been a good data set to see, considering the lack of clarity on reaction time in the method.

Storage conditions for kombucha samples were not noted.

7. Any general comments about the method? * This is a good application of an enzymatic method, and I praise the authors for the work and use of existing instrumentation that is relatively inexpensive and does not require a high level of analyst expertise to run it.

Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale. * If the above mentioned issues can be addressed, I would lean toward recommending the method for First Action

AOAC Stakeholder Panel on Strategic Food Analytical Methods: Expert Review Panel AOAC Candidate Method #KOM-07 Enzytec™ Liquid Ethanol

• Author(s): Markus Lacorn and Thomas Hektor

• Submitted by: Patricia Meinhardt, R-Biopharm, Inc.

• Method applicability: (a) Target Analyte.—Ethanol; CAS Registry No. 64-17-5

(b) Matrices.-- For this validation, Enzytec™ Liquid Ethanol was tested with the following matrices: kombucha, fruit juice, vegetable juice, alcohol-free beer. (c) Claims.-- The Enzytec™ Liquid Ethanol quantifies ethanol in diluted or undiluted samples between 30 and 300 mg/l ethanol with a high precision (CV ≤ 2%). The linear range of the system is between 3.3 and 500 mg/l. The LoD is at 1.9 mg/l. All chemically related primary, secondary, and tertiary alcohols show side-chain activity with exception of methanol. Acetaldehyde interferes at concentrations higher than 3000 mg/l while sulphite interferes at concentrations higher than 300 mg/l. (d) Expression of results.—Results are expressed as g/l ethanol; conversion to %ABV is possible

• Attachments: 3

Submitter notes: We have included additionalmatrices: kombucha, fruit juice, vegetable juice, alcohol- free beer. Primary reviewer: George Joseph, AsureQuality, New Zealand Secondary Reviewer: Armen Mirzoian, US Alcohol & Tobacco Tax & Trade Bureau

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